Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

The deleterious effect of mechanical dissociation of rat testes on the functional activity and purification of Leydig cells using Percoll gradients.

The effect of mechanically dispersing rat testes prior to collagenase treatment on the activity of the subsequently isolated Leydig cells has been investigated. The latter were obtained by centrifugation of the testes cells on Percoll density gradients. It was found that the lutropin-stimulated testosterone and cyclic AMP production were 5 and 3 times higher, respectively, in the purified Leydig cells when the mechanical dispersion was omitted, whereas the specific binding of human choriogonadotrophin was unchanged. It is concluded that mechanical treatment of rat testes, unlike mouse testes, decrease the activity of the Leydig cells.

The effect of cell damage on the density and steroidogenic capacity of rat testis Leydig cells, using an NADH exclusion test for determination of viability.

The ability of cells to exclude pyridine nucleotides was found to be a more sensitive index of cell viability than trypan blue. The three nucleated cell bands obtained by centrifugation of crude rat testis cells on Percoll density gradients contained different proportions of viable and damaged cells. Band III, the steroidogenically active Leydig cell band was mainly viable cells, whilst bands I and II contained 70 and 35% damaged cells respectively. Leydig cells damaged by freezing were shown to have a lower density than viable Leydig cells and did not produce testosterone after stimulation with LH. It is concluded that the methods used to prepare and purify rat testis Leydig cells contribute to the apparent Leydig cell "heterogeneity".

11. Testicular and epididymal function. Effects of isolation and purification procedures on the viability and properties of testis Leydig cells.

The effect of the isolation and purification procedures used to obtain mouse and rat Leydig cells on their viability and steroidgenic properties has been investigated. It was found that mechanical dispersion of rat testes prior to collagenase dispersion had a deleterious effect on the steroidogenic capacity of the cells. Omission of the mechanical disruption and separation of the collagenase-dispersed cells on Percoll gradients yielded 3 bands of cells. Band III (density 1.070 g/ml) contained highly pure viable Leydig cells of high steroidogenic capacity. Diaphorase histochemistry (but not trypan blue exclusion) revealed that bands I and II contained high amounts of damaged Leydig cells. These results are discussed in relation to the previously reported heterogeneity of Leydig cells. Both mouse and rat Leydig cells (band III) in monolayer culture maintained their responsiveness to LH for several days. The rat (but not mouse) Leydig cells contained LHRH receptors and responded to LHRH in a biphasic manner: initially a stimulation of both basal and LH increased steroidogenesis occurred which after 24 h became inhibitory.